NOS1AP variant associated with incidence of type 2 diabetes in calcium channel blocker users in the Atherosclerosis Risk in Communities (ARIC) study.

AIMS/HYPOTHESIS: To validate the reported association between rs10494366 in NOS1AP (the gene encoding nitric oxide synthase-1 adaptor protein) and the incidence of type 2 diabetes in calcium channel blocker (CCB) users and to identify additional NOS1AP variants associated with type 2 diabetes risk. METHODS: Data from 9 years of follow-up in 9,221 middle-aged white and 2,724 African-American adults free of diabetes at baseline from the Atherosclerosis Risk in Communities study were analysed. Nineteen NOS1AP variants were examined for associations with incident diabetes and fasting glucose levels stratified by baseline CCB use. RESULTS: Prevalence of CCB use at baseline was 2.7% (n = 247) in whites and 2.3% (n = 72) in African-Americans. Among white CCB users, the G allele of rs10494366 was associated with lower diabetes incidence (HR 0.57, 95% CI 0.35-0.92, p = 0.016). The association was marginally significant after adjusting for age, sex, obesity, smoking, alcohol use, physical activity, hypertension, heart rate and electrocardiographic QT interval (HR 0.63, 95% CI 0.38-1.04, p = 0.052). rs10494366 was associated with lower average fasting glucose during follow-up (p = 0.037). No other variants were associated with diabetes risk in CCB users after multiple-testing correction. No associations were observed between any NOS1AP variant and diabetes development in non-CCB users. NOS1AP variants were not associated with diabetes risk in either African-American CCB users or non-CCB users. CONCLUSIONS/INTERPRETATION: We have independently replicated the association between rs10494366 in NOS1AP and incident diabetes among white CCB users. Further exploration of NOS1AP variants and type 2 diabetes and functional studies of NOS1AP in type 2 diabetes pathology is warranted.

Mechanism of alpha-adrenergic regulation of expressed hKv4.3 currents.

The transient outward potassium current (I(to)) is an important repolarizing current in the mammalian heart. I(to) is regulated by adrenergic stimulation; however, the effect of agonists on this current, and consequently the action potential duration and profile, is variable. An important source of the variability is the difference in the channel genes that underlie I(to). There are two subfamilies of candidate genes that are likely to encode I(to) in the mammalian heart: Kv4 and Kv1.4; the predominance of either gene is a function of the species, stage of development, and region of the heart. The existence of different isoforms of the Kv4 family (principally Kv4.2 or Kv4.3) further complicates the effect of alpha-adrenergic modulation of cardiac I(to). In the human ventricle, hKv4.3 is the predominant gene underlying I(to). Two splice variants of human Kv4.3 (hKv4.3) are present in the human ventricle; the longer splice variant contains a 19-amino acid insert in the COOH-terminus with a consensus protein kinase C (PKC) site. We used heterologous expression of hKv4.3 splice variants and studies of human ventricular myocytes to demonstrate that alpha-adrenergic modulation of I(to) occurs through a PKC signaling pathway and that only the long splice variant (hKv4.3-L) is modulated via this pathway. Only a single hKv4.3-L monomer in the tetrameric I(to) channel is required to confer sensitivity to phenylephrine (PE). Mutation of the PKC site in hKv4.3-L eliminates alpha-adrenergic modulation of the hKv4.3-encoded current. The similar, albeit less robust, modulation of human ventricular I(to) by PE suggests that hKv4.3-L is expressed in a functional form in the human heart.

Molecular architecture of the voltage-dependent Na channel: functional evidence for alpha helices in the pore.

The permeation pathway of the Na channel is formed by asymmetric loops (P segments) contributed by each of the four domains of the protein. In contrast to the analogous region of K channels, previously we (Yamagishi, T., M. Janecki, E. Marban, and G. Tomaselli. 1997. Biophys. J. 73:195-204) have shown that the P segments do not span the selectivity region, that is, they are accessible only from the extracellular surface. The portion of the P-segment NH(2)-terminal to the selectivity region is referred to as SS1. To explore further the topology and functional role of the SS1 region, 40 amino acids NH(2)-terminal to the selectivity ring (10 in each of the P segments) of the rat skeletal muscle Na channel were substituted by cysteine and expressed in tsA-201 cells. Selected mutants in each domain could be blocked with high affinity by externally applied Cd(2)+ and were resistant to tetrodotoxin as compared with the wild-type channel. None of the externally applied sulfhydryl-specific methanethiosulfonate reagents modified the current through any of the mutant channels. Both R395C and R750C altered ionic selectivity, producing significant increases in K(+) and NH(4)(+) currents. The pattern of side chain accessibility is consistent with a pore helix like that observed in the crystal structure of the bacterial K channel, KcsA. Structure prediction of the Na channel using the program PHDhtm suggests an alpha helix in the SS1 region of each domain channel. We conclude that each of the P segments undergoes a hairpin turn in the permeation pathway, such that amino acids on both sides of the putative selectivity filter line the outer mouth of the pore. Evolutionary conservation of the pore helix motif from bacterial K channels to mammalian Na channels identifies this structure as a critical feature in the architecture of ion selective pores.

Cardiac phosphodiesterase 5 (cGMP-specific) modulates beta-adrenergic signaling in vivo and is down-regulated in heart failure.

Recent studies implicate increased cGMP synthesis as a postreceptor contributor to reduced cardiac sympathetic responsiveness. Here we provide the first evidence that modulation of this interaction by cGMP-specific phosphodiesterase PDE5A is also diminished in failing hearts, providing a novel mechanism for blunted beta-adrenergic signaling in this disorder. In normal conscious dogs chronically instrumented for left ventricular pressure-dimension analysis, PDE5A inhibition by EMD82639 had modest basal effects but markedly blunted dobutamine-enhanced systolic and diastolic function. In failing hearts (tachypacing model), however, EMD82639 had negligible effects on either basal or dobutamine-stimulated function. Whole myocardium from failing hearts had 50% lower PDE5A protein expression and 30% less total and EMD92639-inhibitable cGMP-PDE activity. Although corresponding myocyte protein and enzyme activity was similar among groups, the proportion of EMD82639-inhibitable activity was significantly lower in failure cells. Immunohistochemistry confirmed PDE5A expression in both the vasculature and myocytes of normal and failing hearts, but there was loss of z-band localization in failing myocytes that suggested altered intracellular localization. Thus, PDE5A regulation of cGMP in the heart can potently modulate beta-adrenergic stimulation, and alterations in enzyme localization and reduced synthesis may blunt this pathway in cardiac failure, contributing to dampening of the beta-adrenergic response.

Latent specificity of molecular recognition in sodium channels engineered to discriminate between two "indistinguishable" mu-conotoxins.

mu-Conotoxins (mu-CTX) are potent oligopeptide blockers of sodium channels. The best characterized forms of mu-CTX, GIIIA and GIIIB, have similar primary and three-dimensional structures and comparable potencies (IC(50) approximately 30 nM) for block of wild-type skeletal muscle Na(+) channels. The two toxins are thus considered to be indistinguishable by their target channels. We have found mutations in the domain II pore region (D762K and E765K) that decrease GIIIB blocking affinity approximately 200-fold, but reduce GIIIA affinity by only approximately 4-fold, compared with wild-type channels. Synthetic mu-CTX GIIIA mutants reveal that the critical residue for differential recognition is at position 14, the site of the only charge difference between the two toxin isoforms. Therefore, engineered Na(+) channels, but not wild-type channels, can discriminate between two highly homologous conotoxins. Latent specificity of toxin-channel interactions, such as that revealed here, is a principle worthy of exploitation in the design and construction of improved biosensors.

A functional assay for paralytic shellfish toxins that uses recombinant sodium channels.

Saxitoxin (STX) and its derivatives are highly toxic natural compounds produced by dinoflagellates commonly present in marine phytoplankton. During algal blooms ("red tides"), shellfish accumulate saxitoxins leading to paralytic shellfish poisoning (PSP) in human consumers. PSP is a consequence of the high-affinity block of voltage-dependent Na channels in neuronal and muscle cells. PSP poses a significant public health threat and an enormous economic challenge to the shellfish industry worldwide. The standard screening method for marine toxins is the mouse mortality bioassay that is ethically problematic, costly and time-consuming. We report here an alternative, functional assay based on electrical recordings in cultured cells stably expressing a PSP target molecule, the STX-sensitive skeletal muscle Na channel. STX-equivalent concentration in the extracts was calibrated by comparison with purified STX, yielding a highly significant correlation (R=0.95; N=30) between electrophysiological determinations and the values obtained by conventional methods. This simple, economical, and reproducible assay obviates the need to sacrifice millions of animals in mandatory paralytic shellfish toxin screening programs.

Clockwise domain arrangement of the sodium channel revealed by (mu)-conotoxin (GIIIA) docking orientation.

mu-Conotoxins (mu-CTXs) specifically inhibit Na(+) flux by occluding the pore of voltage-gated Na(+) channels. Although the three-dimensional structures of mu-CTXs are well defined, the molecular configuration of the channel receptor is much less certain; even the fundamental question of whether the four homologous Na(+) channel domains are arranged in a clockwise or counter-clockwise configuration remains unanswered. Residues Asp(762) and Glu(765) from domain II and Asp(1241) from domain III of rat skeletal muscle Na(+) channels are known to be critical for mu-CTX binding. We probed toxin-channel interactions by determining the potency of block of wild-type, D762K, E765K, and D1241C channels by wild-type and point-mutated mu-CTXs (R1A, Q14D, K11A, K16A, and R19A). Individual interaction energies for different toxin-channel pairs were quantified from the half-blocking concentrations using mutant cycle analysis. We find that Asp(762) and Glu(765) interact strongly with Gln(14) and Arg(19) but not Arg(1) and that Asp(1241) is tightly coupled to Lys(16) but not Arg(1) or Lys(11). These newly identified toxin-channel interactions within adjacent domains, interpreted in light of the known asymmetric toxin structure, fix the orientation of the toxin with respect to the channel and reveal that the four internal domains of Na(+) channels are arranged in a clockwise configuration as viewed from the extracellular surface.

Electrical and structural remodeling of the failing ventricle.

Heart failure (HF) is a complex disease that presents a major public health challenge to Western society. The prevalence of HF increases with age in the elderly population, and the societal disease burden will increase with prolongation of life expectancy. HF is initially characterized by an adaptive increase of neurohumoral activation to compensate for reduction of cardiac output. This leads to a combination of neurohumoral activation and mechanical stress in the failing heart that trigger a cascade of maladaptive electrical and structural events that impair both the systolic and diastolic function of the heart.

Probing the interaction between inactivation gating and Dd-sotalol block of HERG.

Potassium channels encoded by HERG underlie I:(Kr), a sensitive target for most class III antiarrhythmic drugs, including methanesulfonanilides such as Dd-sotalol. Recently it was shown that these drugs are trapped in the channel as it closes during hyperpolarization. At the same time, HERG channels rapidly open and inactivate when depolarized, and methanesulfonanilide block is known to develop in a use-dependent manner, suggesting a potential role for inactivation in drug binding. However, the role of HERG inactivation in class III drug action is uncertain: pore mutations that remove inactivation reduce block, yet many of these mutations also modify the channel permeation properties and could alter drug affinity through gating-independent mechanisms. In the present study, we identify a definitive role for inactivation gating in Dd-sotalol block of HERG, using interventions complementary to mutagenesis. These interventions (addition of extracellular Cd(2+), removal of extracellular Na(+)) modify the voltage dependence of inactivation but not activation. In normal extracellular solutions, block of HERG current by 300 micromol/L Dd-sotalol reached 80% after a 10-minute period of repetitive depolarization to +20 mV. Maneuvers that impeded steady-state inactivation also reduced Dd-sotalol block of HERG: 100 micromol/L Cd(2+) reduced steady-state block to 55% at +20 mV (P:<0.05); removing extracellular Na(+) reduced block to 44% (P:<0.05). An inactivation-disabling mutation (G628C-S631C) reduced Dd-sotalol block to only 11% (P:<0.05 versus wild type). However, increasing the rate of channel inactivation by depolarizing to +60 mV reduced Dd-sotalol block to 49% (P:<0.05 versus +20 mV), suggesting that the drug does not primarily bind to the inactivated state. Coexpression of MiRP1 with HERG had no effect on inactivation gating and did not modify Dd-sotalol block. We postulate that Dd-sotalol accesses its receptor in the open pore, and the drug-receptor interaction is then stabilized by inactivation. Whereas deactivation traps the bound methanesulfonanilide during hyperpolarization, we propose that HERG inactivation stabilizes the drug-receptor interaction during membrane depolarization.

Role of the calcium-independent transient outward current I(to1) in shaping action potential morphology and duration.

The Kv4.3-encoded current (I:(Kv4.3)) has been identified as the major component of the voltage-dependent Ca(2+)-independent transient outward current (I:(to1)) in human and canine ventricular cells. Experimental evidence supports a correlation between I:(to1) density and prominence of the phase 1 notch; however, the role of I:(to1) in modulating action potential duration (APD) remains unclear. To help resolve this role, Markov state models of the human and canine Kv4.3- and Kv1.4-encoded currents at 35 degrees C are developed on the basis of experimental measurements. A model of canine I:(to1) is formulated as the combination of these Kv4.3 and Kv1.4 currents and is incorporated into an existing canine ventricular myocyte model. Simulations demonstrate strong coupling between L-type Ca(2+) current and I:(Kv4.3) and predict a bimodal relationship between I:(Kv4.3) density and APD whereby perturbations in I:(Kv4.3) density may produce either prolongation or shortening of APD, depending on baseline I:(to1) current level.

A structural rearrangement in the sodium channel pore linked to slow inactivation and use dependence.

Voltage-gated sodium (Na(+)) channels are a fundamental target for modulating excitability in neuronal and muscle cells. When depolarized, Na(+) channels may gradually enter long-lived, slow-inactivated conformational states, causing a cumulative loss of function. Although the structural motifs that underlie transient, depolarization-induced Na(+) channel conformational states are increasingly recognized, the conformational changes responsible for more sustained forms of inactivation are unresolved. Recent studies have shown that slow inactivation components exhibiting a range of kinetic behavior (from tens of milliseconds to seconds) are modified by mutations in the outer pore P-segments. We examined the state-dependent accessibility of an engineered cysteine in the domain III, P-segment (F1236C; rat skeletal muscle) to methanethiosulfonate-ethylammonium (MTSEA) using whole-cell current recordings in HEK 293 cells. F1236C was reactive with MTSEA applied from outside, but not inside the cell, and modification was markedly increased by depolarization. Depolarized F1236C channels exhibited both intermediate (I(M); tau approximately 30 ms) and slower (I(S); tau approximately 2 s) kinetic components of slow inactivation. Trains of brief, 5-ms depolarizations, which did not induce slow inactivation, produced more rapid modification than did longer (100 ms or 6 s) pulse widths, suggesting both the I(M) and I(S) kinetic components inhibit depolarization-induced MTSEA accessibility of the cysteine side chain. Lidocaine inhibited the depolarization-dependent sulfhydryl modification induced by sustained (100 ms) depolarizations, but not by brief (5 ms) depolarizations. We conclude that competing forces influence the depolarization-dependent modification of the cysteine side chain: conformational changes associated with brief periods of depolarization enhance accessibility, whereas slow inactivation tends to inhibit the side chain accessibility. The findings suggest that slow Na(+) channel inactivation and use-dependent lidocaine action are linked to a structural rearrangement in the outer pore.

Molecular aspects of arrhythmias associated with cardiomyopathies.

The increased risk of sudden cardiac death in patients with myocardial hypertrophy and heart failure is the result of remodeling that occurs in both the myocyte and interstitial compartments of the heart. Action potential prolongation is a hallmark of hypertrophied and failing myocardium and is a consequence of differential expression and function of membrane currents and transporters. Functional downregulation of K currents in the ventricle is a recurring theme in hypertrophy and failure; the reduction in the density of the transient outward current (I(to)) is the most consistent observation, whereas data on the density of the inward (I(K1)) and the delayed rectifier (I(K)) currents are more contradictory. The altered intracellular Ca handling of the myopathic hearts prolongs the decay of the L-type Ca current and favors extrusion of cytosolic Ca2+ via the Na+-Ca2+ exchanger. The interaction between such altered membrane currents and a changed neurohumoral milieu creates a substrate that is highly susceptible to potentially lethal ventricular arrhythmias.

Novel structural determinants of mu-conotoxin (GIIIB) block in rat skeletal muscle (mu1) Na+ channels.

mu-Conotoxin (mu-CTX) specifically occludes the pore of voltage-dependent Na(+) channels. In the rat skeletal muscle Na(+) channel (mu1), we examined the contribution of charged residues between the P loops and S6 in all four domains to mu-CTX block. Conversion of the negatively charged domain II (DII) residues Asp-762 and Glu-765 to cysteine increased the IC(50) for mu-CTX block by approximately 100-fold (wild-type = 22.3 +/- 7.0 nm; D762C = 2558 +/- 250 nm; E765C = 2020 +/- 379 nm). Restoration or reversal of charge by external modification of the cysteine-substituted channels with methanethiosulfonate reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA)) did not affect mu-CTX block (D762C: IC(50, MTSEA+) = 2165.1 +/- 250 nm; IC(50, MTSES-) = 2753.5 +/- 456.9 nm; E765C: IC(50, MTSEA+) = 2200.1 +/- 550.3 nm; IC(50, MTSES-) = 3248.1 +/- 2011.9 nm) compared with their unmodified counterparts. In contrast, the charge-conserving mutations D762E (IC(50) = 21.9 +/- 4.3 nm) and E765D (IC(50) = 22.0 +/- 7.0 nm) preserved wild-type blocking behavior, whereas the charge reversal mutants D762K (IC(50) = 4139.9 +/- 687.9 nm) and E765K (IC(50) = 4202.7 +/- 1088.0 nm) destabilized mu-CTX block even further, suggesting a prominent electrostatic component of the interactions between these DII residues and mu-CTX. Kinetic analysis of mu-CTX block reveals that the changes in toxin sensitivity are largely due to accelerated toxin dissociation (k(off)) rates with little changes in association (k(on)) rates. We conclude that the acidic residues at positions 762 and 765 are key determinants of mu-CTX block, primarily by virtue of their negative charge. The inability of the bulky MTSES or MTSEA side chain to modify mu-CTX sensitivity places steric constraints on the sites of toxin interaction.

Lidocaine induces a slow inactivated state in rat skeletal muscle sodium channels.

1. Local anaesthetics such as lidocaine (lignocaine) interact with sodium channels in a manner that is exquisitely sensitive to the voltage-dependent conformational state of the ion channel. When depolarized in the presence of lidocaine, sodium channels assume a long-lived quiescent state. Although studies over the last decade have localized the lidocaine receptor to the inner aspect of the aqueous pore, the mechanistic basis of depolarization-induced 'use-dependent' lidocaine block remains uncertain. 2. Recent studies have shown that lowering the extracellular Na+ concentration ([Na+]o) and mutations in the sodium channel outer P-loop modulate occupancy of a quiescent 'slow' inactivated state with intermediate kinetics (termed IM) that involves structural rearrangements in the outer pore. 3. Site-directed mutagenesis and ion-replacement experiments were performed using voltage-clamped Xenopus oocytes and cultured (HEK-293) cells expressing wild-type and mutant rat skeletal muscle (mu1) sodium channels. 4. Our results show that lowering [Na+]o potentiates use-dependent lidocaine block. The effect of [Na+]o is maintained despite a III-IV linker mutation that partially disrupts fast inactivation (F1304Q). In contrast, the effect of lowering [Na+]o on lidocaine block is reduced by a P-loop mutation (W402A) that limits occupancy of IM. 5. Our findings are consistent with a simple allosteric model where lidocaine binding induces channels to occupy a native slow inactivated state that is inhibited by [Na+]o.

A revised view of cardiac sodium channel "blockade" in the long-QT syndrome.

Mutations in SCN5A, encoding the cardiac sodium (Na) channel, are linked to a form of the congenital long-QT syndrome (LQT3) that provokes lethal ventricular arrhythmias. These autosomal dominant mutations disrupt Na channel function, inhibiting channel inactivation, thereby causing a sustained ionic current that delays cardiac repolarization. Sodium channel-blocking antiarrhythmics, such as lidocaine, potently inhibit this pathologic Na current (I(Na)) and are being evaluated in patients with LQT3. The mechanism underlying this effect is unknown, although high-affinity "block" of the open Na channel pore has been proposed. Here we report that a recently identified LQT3 mutation (R1623Q) imparts unusual lidocaine sensitivity to the Na channel that is attributable to its altered functional behavior. Studies of lidocaine on individual R1623Q single-channel openings indicate that the open-time distribution is not changed, indicating the drug does not block the open pore as proposed previously. Rather, the mutant channels have a propensity to inactivate without ever opening ("closed-state inactivation"), and lidocaine augments this gating behavior. An allosteric gating model incorporating closed-state inactivation recapitulates the effects of lidocaine on pathologic I(Na). These findings explain the unusual drug sensitivity of R1623Q and provide a general and unanticipated mechanism for understanding how Na channel-blocking agents may suppress the pathologic, sustained Na current induced by LQT3 mutations.

Isoform-specific lidocaine block of sodium channels explained by differences in gating.

When depolarized from typical resting membrane potentials (V(rest) approximately -90 mV), cardiac sodium (Na) currents are more sensitive to local anesthetics than brain or skeletal muscle Na currents. When expressed in Xenopus oocytes, lidocaine block of hH1 (human cardiac) Na current greatly exceeded that of mu1 (rat skeletal muscle) at membrane potentials near V(rest), whereas hyperpolarization to -140 mV equalized block of the two isoforms. Because the isoform-specific tonic block roughly parallels the drug-free voltage dependence of channel availability, isoform differences in the voltage dependence of fast inactivation could underlie the differences in block. However, after a brief (50 ms) depolarizing pulse, recovery from lidocaine block is similar for the two isoforms despite marked kinetic differences in drug-free recovery, suggesting that differences in fast inactivation cannot entirely explain the isoform difference in lidocaine action. Given the strong coupling between fast inactivation and other gating processes linked to depolarization (activation, slow inactivation), we considered the possibility that isoform differences in lidocaine block are explained by differences in these other gating processes. In whole-cell recordings from HEK-293 cells, the voltage dependence of hH1 current activation was approximately 20 mV more negative than that of mu1. Because activation and closed-state inactivation are positively coupled, these differences in activation were sufficient to shift hH1 availability to more negative membrane potentials. A mutant channel with enhanced closed-state inactivation gating (mu1-R1441C) exhibited increased lidocaine sensitivity, emphasizing the importance of closed-state inactivation in lidocaine action. Moreover, when the depolarization was prolonged to 1 s, recovery from a "slow" inactivated state with intermediate kinetics (I(M)) was fourfold longer in hH1 than in mu1, and recovery from lidocaine block in hH1 was similarly delayed relative to mu1. We propose that gating processes coupled to fast inactivation (activation and slow inactivation) are the key determinants of isoform-specific local anesthetic action.

Charged residues between the selectivity filter and S6 segments contribute to the permeation phenotype of the sodium channel.

The deep regions of the Na(+) channel pore around the selectivity filter have been studied extensively; however, little is known about the adjacent linkers between the P loops and S6. The presence of conserved charged residues, including five in a row in domain III (D-III), hints that these linkers may play a role in permeation. To characterize the structural topology and function of these linkers, we neutralized the charged residues (from position 411 in D-I and its homologues in D-II, -III, and -IV to the putative start sites of S6) individually by cysteine substitution. Several cysteine mutants displayed enhanced sensitivities to Cd(2+) block relative to wild-type and/or were modifiable by external sulfhydryl-specific methanethiosulfonate reagents when expressed in TSA-201 cells, indicating that these amino acids reside in the permeation pathway. While neutralization of positive charges did not alter single-channel conductance, negative charge neutralizations generally reduced conductance, suggesting that such charges facilitate ion permeation. The electrical distances for Cd(2+) binding to these residues reveal a secondary "dip" into the membrane field of the linkers in domains II and IV. Our findings demonstrate significant functional roles and surprising structural features of these previously unexplored external charged residues.

Cellular basis of ventricular arrhythmias and abnormal automaticity in heart failure.

The high incidence of sudden death in heart failure may reflect an increased propensity to abnormal repolarization and long Q-T interval-related arrhythmias. If so, cells from failing hearts would logically be expected to exhibit a heightened susceptibility to early afterdepolarizations (EAD). We found that midmyocardial ventricular cells isolated from dogs with pacing-induced heart failure exhibited an increased action potential duration and many more EAD than cells from nonpaced controls; this was the case both under basal conditions (P < 0.01) and after lowering external K(+) concentration ([K(+)](o)) to 2 mM and exposing cells to cesium (3 mM; P < 0.05). An unexpected finding was the occurrence of spontaneous depolarizations (SD, >5 mV) from the resting potential that were not coupled to prior action potentials. These SD were observed in 20% of failing cells (n = 5 of 25) under basal ionic conditions but in none of the normal cells (n = 0 of 27, P < 0.05). The net inward current that underlies SD is not triggered by Ca(2+) oscillations and thus differs fundamentally from the currents that underlie delayed afterdepolarizations. We conclude that cardiomyopathic canine ventricular cells are intrinsically predisposed to EAD and SD. Because EAD have been linked to the pathogenesis of torsade de pointes, our results support the hypothesis that sudden death in heart failure often arises from abnormalities of repolarization. The frequent occurrence of SD points to a novel cellular mechanism for abnormal automaticity in heart failure.

Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, I: experimental studies.

Pacing-induced heart failure in the dog recapitulates many of the electrophysiological and hemodynamic abnormalities of the human disease; however, the mechanisms underlying altered Ca2+ handling have not been investigated in this model. We now show that left ventricular midmyocardial myocytes isolated from dogs subjected to 3 to 4 weeks of rapid pacing have prolonged action potentials and Ca2+ transients with reduced peaks, but durations approximately 3-fold longer than controls. To discriminate between action potential effects on Ca2+ kinetics and direct changes in Ca2+ regulatory processes, voltage-clamp steps were used to examine the time constant for cytosolic Ca2+ removal (tauCa). tauCa was prolonged by just 35% in myocytes from failing hearts after fixed voltage steps in physiological solutions (tauCa control, 216+/-25 ms, n=17; tauCa failing, 292+/-23 ms, n=22; P<0.05), but this difference was markedly accentuated when Na+/Ca2+ exchange was eliminated (tauCa control, 282+/-30 ms, n=13; tauCa failing, 576+/-83 ms, n=11; P<0. 005). Impaired sarcoplasmic reticular (SR) Ca2+ uptake and a greater dependence on Na+/Ca2+ exchange for cytosolic Ca2+ removal was confirmed by inhibiting SR Ca2+ ATPase with cyclopiazonic acid, which slowed Ca2+ removal more in control than in failing myocytes. beta-Adrenergic stimulation of SR Ca2+ uptake in cells from failing hearts sufficed only to accelerate tauCa to the range of unstimulated controls. Protein levels of SERCA2a, phospholamban, and Na+/Ca2+ exchanger revealed a pattern of changes qualitatively similar to the functional measurements; SERCA2a and phospholamban were both reduced in failing hearts by 28%, and Na+/Ca2+ exchange protein was increased 104% relative to controls. Thus, SR Ca2+ uptake is markedly downregulated in failing hearts, but this defect is partially compensated by enhanced Na+/Ca2+ exchange. The alterations are similar to those reported in human heart failure, which reinforces the utility of the pacing-induced dog model as a surrogate for the human disease.